Short-Chain Fatty Acids (SCFAs) are a product of the fermentation of resistant starches and dietary fibers by the gut microbiota. The most important SCFA are acetate (C2), propionate (C3) and butyrate (C4). These metabolites are formed and absorbed in the colon and then transported through the hepatic vein to the liver. SCFAs are more concentrated in the intestinal lumen than in the serum.Butyrate is largely consumed in the gut epithelium, propionate in the liver and acetate in the periphery.SCFAs act on many cells including components of the immune system and epithelial cells by two main mechanisms: activation of G-protein coupled receptors (GPCRs) and inhibition of histone deacetylase.Considering the association between changes in SCFA concentrations and the development of diseases, methods to quantify these acids in different biological samples are important. In this study, we describe a protocol using gas chromatography to quantify SCFAs in the serum, feces and colonic luminal content.Separation of compounds was performed using a DB-23 column (60 m x 0.25 mm internal diameter [i.d.]) coated with a 0.15 µm thick layer of 80.2% 1-methylnaphatalene. This method has a good linear range (15-10,000 µg/ml). The precision (relative standard deviation [RSD]) is less than 15.0% and the accuracy (error relative [ER]) is within ± 15.0%. The extraction efficiency was higher than 97.0%. Therefore, this is cost effective and reproducible method for SCFA measurement in feces and serum.
Knowing that microbial transformations of compounds play vital roles in the preparation of new derivatives with biological activities, risperidone and its chiral metabolites were determined by capillary electrophoresis and hollow fiber liquid-phase microextraction after a fungal biotransformation study in liquid culture medium. The analytes were extracted from 1 mL liquid culture medium into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 100 mmol/L sodium phosphate buffer pH 3.0 containing 2.0% w/v sulfated-α-CD and carboxymethyl-β-CD 0.5% w/v with a constant voltage of -10 kV. The method was linear over the concentration range of 100-5000 ng/mL for risperidone and 50-5000 ng/mL for each metabolite enantiomer. Within-day and between-day assay precisions and accuracies for all the analytes were studied at three concentration levels, and the values of relative standard deviation and relative error were lower than 15%. The developed method was applied in a pilot biotransformation study employing risperidone as the substrate and the filamentous fungus Mucor rouxii. This study showed that the filamentous fungus was able to metabolize risperidone enantioselectively into its chiral active metabolite, (-)-9-hydroxyrisperidone.
A three-phase hollow-fiber liquid-phase microextraction method for the analysis of rosiglitazone and its metabolites N-desmethyl rosiglitazone and ρ-hydroxy rosiglitazone in microsomal preparations is described for the first time. The drug and metabolites HPLC determination was carried out using an X-Terra RP-18 column, at 22°C. The mobile phase was composed of water, acetonitrile and acetic acid (85:15:0.5, v/v/v) and the detection was performed at 245 nm. The hollow-fiber liquid-phase microextraction procedure was optimized using multifactorial experiments and the following optimal condition was established: sample agitation at 1750 rpm, extraction for 30 min, hydrochloric acid 0.01 mol/L as acceptor phase, 1-octanol as organic phase, and donor phase pH adjustment to 8.0. The recovery rates, obtained by using 1 mL of microsomal preparation, were 47-70%. The method presented LOQs of 50 ng/mL and it was linear over the concentration range of 50-6000 ng/mL, with correlation coefficients (r) higher than 0.9960, for all analytes. The validated method was employed to study the in vitro biotransformation of rosiglitazone using rat liver microsomal fraction.
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